Reaction card and use of same

ABSTRACT

The invention concerns a reaction card ( 1 ) consisting of a body, having a front surface ( 62 ) and a rear surface ( 63 ) defined by an edge ( 68 ), at least one input ( 2, 3, 4  and/or  5 ) and at least one output ( 7  and/or  8 ), connected one ( 2, 3, 4  and/or  5 ) to the other ( 7  and/or  8 ) through a network of channels ( 64 ) constituting at least one reaction path for at least one fluid, the fluid or fluids being directed inside the card ( 1 ) via valves ( 11  to  36  and/or  61 ); each valve ( 11  to  35  or  61 ) consists of a flexible film ( 67 ), which can be deformed to allow a fluid to pass through, the film ( 67 ) being fixed on the rear surface ( 63 ) of said card ( 1 ) at the peripheral indentation of the assembly of channels associated with the valve ( 11  to  35  or  61 ); the card ( 1 ) features channels ( 64 ) flush with at least one of its surfaces ( 62  and/or  63 ), the channels ( 64 ) being of two different cross-sections, a small cross-section serving as a reaction compartment; each front ( 62 ) or rear ( 63 ) is delimited by at least one film ( 48, 49, 65, 66  and/or  67 ).  
     The invention is particularly applicable in the field of diagnosis.

DESCRIPTION

[0001] This invention concerns a reaction card to be used for conductingchemical and/or biological reactions. It also concerns the use of such acard to purify and amplify nucleic acids and to detect them.

[0002] The background art is given in patent U.S. Pat. No. 4,585,623which describes an apparatus for the rapid execution of chemical orimmunochemical assays in a single unit. This apparatus comprises amoulded plastic body (which can be miniaturized) with severalreagent-containing tubes, one tube containing the sample, and another,smaller tube to receive the reaction. Since each tube is fitted with aplunger, the apparatus can be introduced into a programmable machine.

[0003] Even though it can be miniaturized, this apparatus is stillrelatively bulky because space has to be provided for said apparatus aswell as the various connecting rods to drive the plungers. Moreover,only one reaction can be conducted with such an apparatus. If more thanone reaction has to be carried out, the corresponding number ofapparatuses will be needed as well as extra time for loading each withthe appropriate reagents and samples.

[0004] Document WO-A-97/27324 concerns a cassette for conducting severalparallel reactions which includes an inlet opening and an outlet openingfor the transfer of one or more samples to be introduced into thecassette. Some parts of the cassette are constructed in a particular way(the Bursapak chamber, piston valve and ball valve) so that a channel iskept open or closed when pressure is exerted continuously from theinside or outside. The Bursapak chambers are sometimes associated withhydrophobic filters which allow the progression of a fluid to beblocked.

[0005] However, this construction features a major drawback whichresides in the valve structure of the Bursapak chambers. These allinclude films which must be preformed prior to their installation on thereaction card. This pre-forming operation enables each Bursapak chamberto be maintained closed in rest position (FIGS. 2A and 2B). This chambercan be opened by means of an interior action (FIG. 2C), such as, forexample, by an increase in the pressure of a fluid contained in thereaction card, or by means of an exterior action (FIGS. 2D and 2E), suchas an increase in the pressure of a piston or other element against thepreformed film, for example. This preforming of each film must thus beundertaken before it is installed on said reaction card. The cost offabrication, storage and transport of such films is definitely greaterthan for films which remain flat. In addition, this does not facilitatethe fabrication, storage and transport of reaction cards that are soequipped. Finally, the convex shapes of these films can be damaged moreeasily, which can lead to leakage or errors during the subsequent use ofsaid cards.

[0006] Document WO-A-97/02357 describes a nucleic acid diagnosticapparatus which is similar to the previous cassette in terms ofcomplexity. FIG. 2b presents a valve having a great number ofcomponents.

[0007] This apparatus thus essentially has the same drawbacks asWO-A-97/27324.

[0008] Patent application WO-A-99/33559 proposes a card or cartridge forseparating a specific constituent, such as nucleic acids, present in asample. To this end, it features a number of compartments and channels.From the start, these compartments contain all the liquids (elutionliquid, wash solution, for example) that will be used. Fluid movementsare controlled by valves placed irregularly within the card, and byfluid sensors. These valves operate as fluid diodes which allow a fluidto flow in one direction only. To do this, they use magnetic disks whichcan be moved from the outside. The sensors are electrically connected tothe outside or to a microprocessor inside the card.

[0009] This type of card is particularly complex as its body consists ofa sandwich of several stacked elements; refer to the fluid diodes inthis respect. In addition, there must be a hermetic seal between thesevarious superimposed elements. In addition to the sensors, the cardincludes electric wires, fluid diodes operating with magnets, and even amicroprocessor. Fabrication cost is thus prohibitive, and all of theseelectrical and magnetic components can influence the internal operationof said card. A card of this type is thus not very well adapted tomicro-fluid based technology. The presence of fluids inside the cardthus limits storage time or requires that an experienced handlerintroduce these fluids. Operational flexibility is thus rather limited.

[0010] As provided by the invention, a reaction card is proposed whichaddresses all of these problems. Such a card proposes biologicalanalysis of one or more ligands, requiring the use of one or moreanti-ligands for their detection and/or quantification. One example ofan application of the test methods concerns immunoassays, whatever theirparticulars and whether the assay is direct or based on competition.Another application example concerns the detection and/or thequantification of nucleic acids including all operations required forthis detection and/or quantification from any specimen containing thetarget nucleic acids. Among these various operations, reference can bemade to lysis, melting, concentration, purification, the steps ofenzymatic amplification of nucleic acids, the detection stepsincorporating a hybridising step using a DNA chip or a labelled probe,for example. Patent application WO-A-97/02357 or the patent applicationfiled by the applicant under number FR99/00111, clarifies the varioussteps necessary in the case of nucleic acids.

[0011] To this effect, the invention concerns a reaction card consistingof a body, having a front surface and a rear surface defined by an edge,at least one input and at least one output, connected to each otherthrough a network of channels constituting at least one reaction pathfor at least one fluid, the fluid or fluids being directed inside thecard via valves; each valve consists of a flexible film, which can bedeformed to allow a fluid to pass through or which cooperates with acompression means to allow the fluid to pass through, the film beingfixed on the rear surface of said card at a peripheral indentation ofthe assembly of channels associated with the valve; the card compriseschannels flush with at least one of its surfaces, the channels being oftwo different cross-sections, a small cross-section for the transfer offluid or fluids and a large cross-section serving as a reactioncompartment; each front or rear surface is bounded by at least one film.

[0012] According to a specific embodiment, the body of the card ismonobloc and the compression means is affixed and integral with saidcard, or forms a part of an apparatus allowing the card to beimplemented.

[0013] According to a specific embodiment, the ratio between the smallcross-section and the large cross-section of the channels is between1:1.01 and 1:10, preferably between 1:1.01 and 1:3.

[0014] Still according to a specific embodiment, the channels are flushwith all or part of the front surface of the card and the valves arepresent on the rear surface of said card.

[0015] According to yet another specific embodiment, the front surfaceof the card features a single film at the level of all the channelsflush with this surface, and the rear surface of said card features:

[0016] at least a flexible film on the valves,

[0017] at least a hydrophobic filter, and possibly

[0018] at least a film at the level of the channels flush with the rearsurface.

[0019] According to a specific embodiment, the flexible film(s) form asingle film.

[0020] According to another specific embodiment, when the card issubstantially parallelepiped-shaped, the channels are, totally or inpart, circumscribed in the middle part of the card, the blockingfilters, which are hydrophobic, are circumscribed on at least one of thesides of said card, and the valves are positioned between the channelsand the blocking filters.

[0021] In this last embodiment and preferably, the edge includes all ofthe card's fluid inlets and outlets.

[0022] In the case where the card would have a substantiallyparallelepiped shape, the inlet or inlets are located on one of thesides forming the edge, while the outlet or outlets are located on theother side of this edge.

[0023] In this last case and preferably, the inlet or inlets are locatedon a side opposite the side where the outlet or outlets are located.

[0024] In the case where the card is substantially of rectangularparallelepiped shape, the inlet(s) and outlet(s) are located on the twoshort sides forming the edge.

[0025] This invention also concerns the use of a reaction card, asdefined above, for testing a biological solution, possibly pre-treatedto free ligands; the various biological steps are conducted in thefollowing order:

[0026] capture of ligands,

[0027] recovery of captured ligands in an elution solution,

[0028] mixture of recovered ligands with structural and functionalconstituents allowing the ligands to be treated, and

[0029] qualitative and/or quantitative detection of treated ligands.

[0030] This invention also concerns the use of a reaction card, asdefined above, for testing a biological solution, possibly pre-treatedin order to free the nucleic acids; various biological steps are carriedout in the following order:

[0031] capture of nucleic acids,

[0032] recovery of captured nucleic acids in an elution solution,

[0033] mixture of nucleic acids recovered with the structural andfunctional constituents allowing amplification of these nucleic acids,and

[0034] qualitative and/or quantitative detection of amplificationproducts.

[0035] If necessary, a last step is carried out consisting in analysingthese ligands or these amplification products, either in a new locationwithin the card, or after transfer to another apparatus. According to aspecific variant of the use, the ligands or nucleic acids captures aresubjected to at least a wash solution prior to their recovery.

[0036] According to another usage variant, the capture and possiblewashing operations are conducted N times in succession, N being between1 and 10.

[0037] According to yet another specific usage variant, a treatment ofligands or an amplification of nucleic acids is conducted prior to thetransfer to a card compartment or to another apparatus allowing theligands or the amplicons to be analysed.

[0038] Preferably, for all the usage variants described above, the cardis used after an apparatus allowing a test solution to be treated, suchas an apparatus designed to lyse biological cells and to free theligands, such as nucleic acids, and before an apparatus designed toanalyse the presence of these ligands, such as a DNA chip.

[0039] Structurally, the card according to the invention is insertedbetween two devices which can also be cards, one upstream whichpre-treats the test solution, and the other downstream which post-treatsthe solution already tested.

[0040] The accompanying drawings are given by way of example and are notto be taken as limiting in any way. They are designed to make theinvention easier to understand.

[0041]FIG. 1 represents a front view of the reaction card according to afirst embodiment of the invention.

[0042]FIG. 2 represents an identical although partial view of FIG. 1,when the card's operating process is underway, said card having receivedthe test solution.

[0043]FIG. 3 represents a view identical to FIG. 2, when the card'soperating process is underway, said card having undergone a purgeallowing the volume of the test solution to be precisely metered.

[0044]FIG. 4 represents an identical although partial view of FIG. 1,when the card's operating process is underway, the test solution beingtransferred from the solution's dosing compartment to the part upstreamfrom the separation compartment.

[0045]FIG. 5 represents a view identical to FIGS. 2 and 3, when thecard's operating process is underway, said card having received theelution solution.

[0046]FIG. 6 represents a view identical to FIG. 2, 3 and 5 when thecard's operating process is underway, said card having undergone a purgeallowing the volume of elution fluid to be precisely metered.

[0047]FIG. 7 represents an identical although partial view of FIG. 1,when the card's operating process is underway, the elution fluid beingin the separation chamber and allowing nucleic acids to be recovered.

[0048]FIG. 8 represents an identical although partial view of FIG. 1,when the card's operating process is underway, the elution fluid, whichcontains nucleic acids, being transferred into the structural componentrecovery compartment allowing amplification to take place.

[0049]FIG. 9 represents a view identical to FIG. 8, the elution fluid,which contains nucleic acids and the structural constituents, beingtransferred into the functional constituent recover compartment allowingamplification to take place.

[0050]FIG. 10 represents a view identical to FIGS. 8 and 9, the elutionfluid, which contains the nucleic acids and the functional andstructural constituents, being partly transferred into the samplingcompartment in view of the step represented in FIG. 12.

[0051]FIG. 11 represents a view identical to FIGS. 8 to 10, the elutionfluid, which contains the nucleic acids and the functional andstructural constituents and that was not transferred into the samplingcompartment, being transferred into the convergence compartment, towardsits discharge to the outside.

[0052]FIG. 12 represents a view identical to 8 to 11, the elution fluid,which is present in the sampling compartment, being transferred into thedetection and reading compartment.

[0053]FIG. 13 shows a cross-sectional view through A-A in FIG. 3.

[0054]FIG. 14 represents a front view of the reaction card according toa second embodiment of the invention.

[0055]FIG. 15 represents a perspective view of several valves associatedwith the reaction card.

[0056] Finally, FIG. 16 represents a cross-sectional view along B-B ofFIG. 15.

DEFINITIONS

[0057] The term ligand refers to all biological species which can be ofnucleic acid or proteic nature, such as an antigen, an antigen fragment,an antibody, an antibody fragment, a hapten, a nucleic acid, a nucleicacid fragment, a vitamin, a peptide or a polypeptide, for example.

[0058] A pre-treatment example could consist of a cellular lysis, suchas described in the applicant's patent applications:

[0059] FR99/04289, filed Apr. 1, 1999, about lysis by sonication,

[0060] PCT/FR99/01309, filed under priority Jul. 23, 1998, about mixedmagnetic and mechanical lysis,

[0061] PCT/FR99/00830, filed under priority Apr. 10, 1998, aboutelectric lysis,

[0062] PCT/IB98/01475, filed under priority Sep. 23, 1997, aboutmechanical lysis.

[0063] A post-treatment example, may consist of detection using abiochip. The term biochip refers to all solid support on which ligandsare applied, and particularly the term DNA chip refers to all solidsupport on which nucleic acids are applied. The ligand fixation methodcan be performed in various ways and notably, for example, through insitu synthesis by photolithographic techniques or by a piezo-electricsystem, by capillary deposit of preformed ligands. To illustrate this,examples of these biochips applied to DNA chips were given inpublications by G. Ramsay, Nature Biotechnology, 16, p40-44, 1998; F.Ginot, Human Mutation, 10, p1-10, 1997; J. Cheng et al, Moleculardiagnosis, 1(3), p183-200, 1996; T. Livache et al, Nucleic AcidsResearch, 22(15), p2915-2921, 1994; J. Cheng et al, NatureBiotechnology, 16, p541-546, 1998 or in patents U.S. Pat. No. 4,981,783(Augenlicht), U.S. Pat. No. 5,700,637 (Southern), U.S. Pat. No.5,445,934 (Fodor), U.S. Pat. No. 5,744,305 (Fodor), U.S. Pat. No.5,807,522 (Brown).

[0064] The following definition is to be given to “structuralconstituents allowing the amplification when they are associated withthe functional constituents defined below”: nucleotides(desoxyribonucleotides dNTP and/or ribonucleotides NTP), at least onepair of primers which surround specific regions that we wish to amplify,ions (MgCl₂, KCl, for example) and a buffer (such as Tris).

[0065] The functional constituents are formed by at least one enzyme,preferably two or three enzymes, in the presence of a buffer, such asTris at pH 7.5, salts and ions allowing the amplification reaction whenthey are associated with the structural constituents described above.

[0066] The term amplicon is used to refer to products of an enzymaticamplification reaction.

DESCRIPTION OF THE REACTION CARD

[0067] The invention concerns a reaction card 1 represented in a firstembodiment in FIG. 1. This reaction card 1 consists of a front surface62 and a rear surface 63 connected by an edge, also referred to as theside 68. All of the elements which form the front surface 62 in thisfigure are represented in solid lines. In addition, a certain number ofthrough channels 64 can be noted on this surface 62. These channels 64are partitioned by a transparent film 65, affixed on said front surface62. Nonetheless, it is not mandatory that this film 65 be transparent,as those which will be described below; it may be opaque, translucent,etc. In addition, the transparent nature allows better viewing of theposition of the biological solution being tested 69, or any othersolution 70, 71 or 72, introduced into the card 1. The rear surface 63also features a transparent film 66 which partitions the channels 64,which occasionally are flush with the front 63. These films 65 and 66consist of BOPP films (Biaxially Oriented PolyPropylen) or other filmsof the same type, which are soldered or bonded to the body of the card1, this body being inert in relation to the solutions transferred 69 to72 or to the reactions undergone.

[0068] This film 66 may be present on the entire surface of the card 1,or on certain portions of said card 1. Nonetheless, this film 66 mayconsist of a partition made of the same material as the rest of the card1.

[0069] It should also be noted that the front 63 is equipped with acertain number of valves, referenced 11 to 35 and also 61 on the figure;these valves 11 to 35 and 61 correspond to the valves described inpatent application FR99/08116 filed Jun. 22, 1999 in the name of theapplicant. Like this document, all of the valves 11 to 35 and 61 aredefined in relation to the outside of the rear surface 63 by atransparent film 67. This film is sufficiently flexible to allow a testsolution 69, a treated solution 70, a wash solution 71, or an elutionfluid 72, etc. to pass through. It may consist of a silicone membrane ora PE/PET (PolyEthylen/PolyEthylen Tetraphtalate) complex film.

[0070] It should be noted that the transparent films 66 and 67 locatedon the rear surface 63, can be made up of a single transparent film.This would facilitate the fabrication of the card 1. In addition, thetransparent film 65 located on the front 62, and the transparent films66 and 67 located on the rear surface 63, can consist of a singletransparent film, which further facilitates the fabrication of such acard which is so equipped.

[0071] On this rear surface 63, the presence of several hydrophobicfilters 48 and 49 is noted, whose role will be explained below. In fact,in this embodiment of the card in FIG. 1, there are two parallel paths.A left-hand path and a right-hand path which allow the same process tobe conducted. Nonetheless, it is quite possible to have only one path ora plurality of paths, that is more than two, according to the number ofreactions that one wants to perform.

[0072] Finally, on the periphery of the card 1, and more precisely onthe two short sides, inlets and outlets can be noted which are separatedfrom one another. Thus, at the top of FIG. 1, four inlets arerepresented which correspond to four well-defined functions. Thus, ifthe inlet functions are numbered from left to right, the presence of aninlet 2 for the wash solution 71, an inlet 3 for the test solution 69,an inlet 4 for the elution fluid 72, and finally an inlet for thepressure variation 5 can be noted inside said card 1. All of theseinlets 2 to 5 are connected to an inlet ball valve 6 by means of acertain number of channels 64 as well as the first and second valves 11and 12.

[0073] The opposite edge of the card 1 features outlets 7 and 8. Thefirst outlet, outlet 7 for the solution treated 70, is an outletdesigned to discharge a predetermined quantity of the solution treated70 to the outside with a view to subsequent analysis or other subsequenttreatment. The second outlet is an outlet for all biological waste andliquids derived from operation of the card 1. The outlet 7 is associatedwith an outlet ball valve to the outside 9 while the waste outlet isassociated with an outlet ball valve for the wastes 10.

[0074] Besides the two parts, inlets 2 to 5 in the upper position andoutlets 7 and 8 in the lower position, a number of channels, valves andcompartments can be noted between these two extreme parts. As alreadymentioned, the presence of two separate, though identical paths on theleft and right of FIG. 1 can be seen. The first compartment, in theupper position and in the shape of an inverted C on the left-hand pathand in the shape of a C on the right-hand path, is large and consists ofa solution-dosing compartment 36. This compartment 36 contains anelution fluid-dosing compartment 37, which is of similar shape althoughhaving a different volume. The ratio between the volume of thesolution-dosing compartment 36 and the volume of the elutionfluid-dosing compartment 37 is between 1:1 to 10:1, preferably between1.5:1 to 4:1 and yet more preferably 2:1.

[0075] A separation compartment 38 is in underlying position whichallows recovery of capture entities, such as magnetic beads, possiblycoated with capture oligonucleotides, by adsorption or covalence (referto patents U.S. Pat. No. 4,672,040 and 5,750,338), and thus, aftermixture, the selection by separation of nucleic acids that are to beamplified at a later date. The magnetic beads are inside this separationcompartment 38 in the form of a tablet of magnetic beads 39. Theapplicant, under the following references, describes a particularlyinteresting embodiment of these magnetic beads in the patentapplications filed:

[0076] PCT/FR97/00912 under French priority, May 24, 1996, and

[0077] PCT/FR99/00011 under French priority, Jan. 6, 1998.

[0078] The last of these patent applications concerns thermosensitivemagnetic beads, each having a magnetic core covered with an intermediatelayer. The intermediate layer is itself covered by a polymer-based outerlayer capable of interacting with at least one biological molecule. Theouter polymer is thermosensitive and has a predetermined lower criticalsolution temperature (LCST) between 10 and 100° C. and preferablybetween 20 and 60° C. This external layer is synthesized from cationicmonomers, which generate a polymer capable of binding with the nucleicacids. This intermediate layer isolates the core's magnetic charges, inorder to avoid the inhibition problems of the amplification techniquesof these nucleic acids. Magnetic beads in the form of tablets havealready been described in the prior art, formed by EP-A-0.811.694, forexample. The fabrication of tablets in general is also well described inthe prior art, for example U.S. Pat. No. 4,678,812 and U.S. Pat. No.5,275,016. The manufacturing process mentioned above may be used for thesynthesis of other tablets which will be disclosed below.

[0079] A compartment for recovering structural constituents forsubsequent amplification is located in an underlying position. Thiscompartment 40 also contains a tablet 41 consisting of constituentswhich will allow subsequent amplification. Constituents in the form oftablets, to conduct amplification, have already been described in theprior art. Information can be found in the following documents: thepatent U.S. Pat. No. 5,098,893 or the article“Ambient-temperature-stable molecular biology reagents”, R. Ramanujam etal., Product Application Focus, Vol. 14, No. 3 (1993), 470-473, forexample.

[0080] Still in underlying position, an amplification compartment 42 ispresent which features a tablet 43 containing functional constituents,such as enzymes which, when associated with the structural constituentsmentioned previously, will truly lead to an amplification. Enzymes inthe form of tablets, to conduct amplification, have already beendescribed in the prior art. Information can be found in the followingdocuments: patent U.S. Pat. No. 4,891,319, WO-A-87/00196, WO-A-95/33488or the article entitled “Extraordinary stability of enzymes dried intrehalose: simplified molecular biology” by C. Colaco et al.,Bio/Technology, Vol. 10, September 1992, 1007-1011.

[0081] All amplification techniques can be used. As such, the followingnucleic acid amplification techniques exist, among others:

[0082] PCR (Polymerase Chain Reaction), as described in patents U.S.Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159,

[0083] LCR (Ligase Chain Reaction), described in patent applicationEP-A-0.201.184,

[0084] RCR (Repair Chain Reaction), described in patent applicationWO-A-90/01069,

[0085] 3SR (Self Sustained Sequence Replication) with the patentapplication WO-A90/06995,

[0086] NASBA (Nucleic Acid Sequence-Based Amplification) with the patentapplication WO-A-91/02818,

[0087] SPSR (Single Primer Sequence Replication) with patent U.S. Pat.No. 5,194,370, and

[0088] TMA (Transcription Mediated Amplification) with patent U.S. Pat.No. 5,399,491.

[0089] However, it is possible to perform other steps in the compartment42 above. For example, these primers could possibly contain afluorescent marker for the subsequent detection of amplificationproducts, without having to perform an additional step for thislabelling. In this manner, such primers are described in the article byD. Whitcombe et al., “Detection of PCR products using self-probingamplicons and fluorescence”, Nature Biotechnology (17) 1999, pp804, orin the patent applications GB-A-2.338.301, WO-A-99/29905 andWO-A-99/60157.

[0090] An amplification compartment 42 is located below the samplingcompartment 44 which will enable the volume of the treated solution 70,present in said amplification compartment 42, to be divided in two. Thissampling compartment 44 is designed to receive a predetermined quantityof said treated solution 70. The remaining solution 70 is then beingtransferred to a convergence compartment 47 located in the lowerposition of said card 1, but in upper position in relation to the fluidoutlet means which are associated with treated solution 70 or thewastes. The convergence compartment 47 thus contains a single solutioncontaining all the amplified solutions that must either undergo othertreatments within the same card, or be transferred to another apparatusfor analysis. This single solution comes from all of the paths, whichmake up the card 1, that is in this case, the left-hand path and theright-hand path.

[0091] When the amplification products contained in the convergencecompartment 47 are transferred to another apparatus for analysis, thetransfer can be carried out via a tube, as described in FIGS. 8 to 11 ofpatent request PCT/FR99/02137 filed Sep. 8, 1999 by the applicant. Thisother apparatus may consist of a support featuring, on one of its sidesfor example, high density capture probes, DNA chips for example,developed by the Affymetrix Company (“Accessing Genetic Information withHigh-Density DNA arrays”, M. Shee et al., Science, 274, 610-614.“Light-generated oligonucleotide arrays for rapid DNA sequenceanalysis”, A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994,91, 5022-5026), or any other chip system containing nucleic acids fixedon a solid support, such as the DNA chips defined above.

[0092] The aliquot present in the sampling compartment 44 will then betransferred to a detection and reading compartment 45. This compartment45 features a tablet which contains detection means 45 a. Based on theprior art documents mentioned above, it is possible to manufacture, forexample, tablets 45 a containing labelled nucleic acids complementary ofall or part of the amplicons which must be obtained during the previoussteps.

[0093] As described previously, it is also possible to perform thelabelling step during amplification by means of labelled primers. Inthis case, a tablet 45 a is not needed, and the compartment 45 is thussimply a reading compartment.

[0094] This labelling and reading compartment 45 thus features a markertablet 45 a at its inlet and a reading cell 46 downstream. This readingcell 46 allows detecting if amplification has been successful, byfluorescence measurement for example. If so, this allows the technicianor the programmable apparatus, which uses such a card 1, to determine ifthere is need to transfer the treated solution 70, present in theconvergence compartment 47 to amplified sequence analysis means, thesemeans being able to be integrated into the card 1 or contained inanother apparatus more specifically designed for this function.

[0095] The movement of fluids within the card 1, whether it be the testsolution 69, the solution treated 70, the wash solution 71 or theelution fluid 72, are produced through hydrophobic filters which arelocated downstream from certain valves. Each hydrophobic film 48 or 49features a number of blocking filters, referenced 50 to 55 for the film48 or 56 to 60 for the film 49. Their use will be described below.

DESCRIPTION OF THE VALVES

[0096] As described in patent application FR99/08116, filed in 1999 bythe applicant, valves 11 to 35 and 61 have a structure which address theproblems of welding films onto a solid support, such as the body of areaction card 1, generally consisting of plastic materials. Thisinvention offers a particularly interesting application in the card 1according to the invention, as the body of said card 1 features aperipheral groove or reinforcement 74, clearly shown in FIG. 16, whichwill receive a flexible transparent film 67 in the zone circumscribed,said films 67, of which there are four in FIG. 1, and the body of thecard 1 being integral to one another by a weld 84 located in the bottomof the groove 74. As a result, the weld causes no deformation of theupper surface of the card 1 and thus no subsequent problem for usingsaid card 1 and performing assays. The manipulation of electromagnets oractuators of adapted size, as represented in FIGS. 15 and 16, can beused to control these valves 11 to 35 and/or 61.

CARD OPERATION

[0097] In the following description, the reference made to valves 11 to35 and 61 will concern only valves 11 to 35 of the left-hand path, inthe case where the path concerned is not mentioned. All informationconcerning valves 11 to 35 and 61 of the right-hand path will have theirposition on this right-hand path specified. In addition, it should bepointed out that when a valve 11 to 35 is closed, it is either theleft-hand orifice, referred to as the outside orifice, or the right-handorifice, referred to as the inside orifice, of said valve 11 to 35 whichis plugged. This is also true for the right-hand path and for valves 11to 35 and 61, although the position is reversed. Thus, the right-handorifice is referred to as the outside orifice, and the left-hand orificeis referred to as the inner orifice, of said valve 11 to 35 or 61.

[0098] Finally, the valves 11 to 35 and 61 are normally at rest in openposition. During the transfer of fluid, liquid or gas, the closed valvesdirect this fluid.

[0099] 1^(st) step-Dosing of the test solution:

[0100] In this first step, the operation of all the components will bedescribed in detail. While subsequent steps will not be described withthe same detail level, the main functions will be the same, andinformation will be easy to extrapolate.

[0101] The test solution 69 is introduced into the card 1 via the inlet3, along F2. The solution 69 follows the channel 64 between the inlet 3and the first valve 11, which is open. Owing to this, said solution 69can flow to the second valve 12 whose outside orifice is closed, whichprevents this solution 69 from entering the channel 64 which returns tothe inlet 2 and directs said solution 70 to the ball valve of inlet 6which is open. Two channels 64 leave this ball valve 6, one toward theleft-hand path and the other toward the right-hand path. The channelleading to the right-hand path is plugged by the closure of the innerorifice of the second valve 12 of the right-hand path. The channelleading to the left-hand path allows the test solution 69 to passthrough, as the third valve 13 and the seventh valve 17 are open. Theobjective is to allow the dosing compartment 36 to be filled with saidsolution 69 according to FIG. 2. For this reason, the inside valve ofthe forth valve 14 and the outside orifice of the eighth valve 18 areclosed, as is the case of the outside valve of the fourth valve 14 ofthe right-hand path.

[0102] If one wants to also fill the dosing compartment 36 with the testsolution 69 from the right-hand path, the inside orifice of the forthvalve 14 of the right-hand path must be closed, which leaves the outsideorifice of this valve 14 open so that the compartment 36 can be filled,while that of the dosing compartment 37 of the elution liquid 72 is notpossible. The outside orifice of the intermediary valve 61 and theoutside orifice of the intermediate valve 61 and the outside orifice ofthe eighth valve 18 of the right-hand path must be closed.

[0103] Now returning to the single filling of compartment 36 of theleft-hand path, the test solution 69 will be able to fill all thechannels 64 from the inlet 3 to the blocking filter 51 of said solution69. This blocking filter 51 is placed in the continuation of the outsideorifice of the seventh valve 17. It consists of a first hydrophobicfilter 48, which allows gaseous, but not liquid, fluids to pass through.An interesting material is used to make this filter: the VERSAPOR 200Rmembrane manufactured by the American company GELMAN SCIENCES (USA). Itis the configuration of FIG. 2. When this type of filter has been usedonce, it cannot be reused a second time.

[0104] Finally, in order to accurately meter the volume of solution 69in the dosing compartment 36, the volume of which is 100 μl, thechannels 64 must be purged. To do this, a wash solution 71 is introducedvia the inlet 2 along F1. In this case, the following valves are closed:

[0105] the inside orifice of the first valve 11,

[0106] the inside orifice of the third valve 13,

[0107] the inside orifice of the fourth valve 14,

[0108] the inside orifice of the eighth valve 18,

[0109] the inside orifice of the fourteenth valve 24,

[0110] the inside orifice of the fifteenth valve 25,

[0111] the inside orifice of the second valve 12 of the right-hand path,

[0112] the inside orifice of the third valve 13 of the right-hand path,

[0113] the inside orifice of the fourth valve 14 of the right-hand path,

[0114] the inside orifice of the eighth valve 18 of the right-hand path,

[0115] the inside orifice of the fourteenth valve 24 of the right-handpath,

[0116] the inside orifice of the fifteenth valve 25 of the right-handpath,

[0117] Once the washing operation is finished, the wash solution 71present in the channels 64 is replaced by an inert gas, air for example.In this case, air is injected under pressure by the inlet 5 along F4.The closed valves are thus indicated, except for the followingcharacteristics:

[0118] reopening of the inside orifice of the first valve 11,

[0119] closure of the inside orifice of the second valve 12,

[0120] closure of the inside orifice of the first valve 11 of theright-hand path, and

[0121] reopening of the inside orifice of the second valve 12 of theright-hand path.

[0122] In this configuration, the result obtained is that represented inFIG. 3. The test solution 69, which was present at the level of thechannels 64, was thus discharged by the waste outlet 8 along F6. Inaddition, the channels 64 are clean. Only the length of channel 64between the inlet 2 and the inner orifice of the second valve 12contains wash solution 71, which can be used at a later time. Of course,the ball valves of inlet 6 and the waste outlet 10 must be open.

[0123] This washing operation can, of course, be repeated several times.However, this result can be obtained by not performing the washingoperation. In this case, the remaining solution 69 located in thechannels 64 only has to be pushed via the air inlet 5 along F4. Theresult will be identical except there will be no wash solution 71between the inlet 2 and the outside orifice of the second valve 12, andthe wash solution 71 present between the inside orifice and the secondvalve 12 and the ball valve of inlet 6 will be replaced by the testsolution 69. This possibility is thus less interesting, even if itspossible, as solution 69 likely to pass the ball valve 6 remains in thecard 1 as soon as wash solution 71 is introduced along F1.

[0124] In both cases proposed, the nucleic acids in this compartment 36can be denatured. To do this, it is heated to a temperature between 80and 100° C.

[0125] 2^(nd) step-Separation of the nucleic acids contained in the testsolution:

[0126] In this second step, the test solution 69, which has just beendosed and is present in the compartment 36, is transferred in thefollowing compartment, referred to as the separation compartment 38. Thetest solution 69 located in compartment 36 is thus pushed toward theseparation compartment 38, by the introduction of an inert fluid intothe channel network 64 via the input 5 along F4. In this case, thefollowing valves are closed:

[0127] the inside orifice of the second valve 12,

[0128] the inside orifice of the fourth valve 14,

[0129] one of the orifices of the sixth valve 16,

[0130] the inside orifice of the eighth valve 18,

[0131] the inside orifice of the eleventh valve 21,

[0132] the outside orifice of the fifteenth valve 25,

[0133] the inside orifice of the first valve 11 of the right-hand path,

[0134] the outside orifice of the fourth valve 14 of the right-handpath,

[0135] For informational purposes, the closing of a valve cannot bemandatory, if the closure of another upstream or downstream valve hasthe same effect. In this manner, the sixth valve 16 can remain open. Inthis case, the inside orifice of the fifth valve 15 should be closed.

[0136] The transfer of the dosed solution 69 is obtained as clearlyrepresented in FIG. 4 and is made possible by the presence in relationwith this compartment 38 with a blocking filter 52 which is connected toa branch connection 73 in intermediate position along said compartment38. This filter 52 being associated with the tenth valve 20, the airdownstream of said solution 69 can escape via this blocking filter 52.When the solution 69 reaches the blocking filter 52, via the branchconnection 73, the progress of said solution 69 is stopped. Furthermore,the total volume of this solution 69 is less than the volume of saidcompartment 38 upstream from said branch connection 73 and shall thus becircumscribed in this upstream part of the compartment 38.

[0137] It should be noted that in this position, the dosed solution 69has not yet reached the magnetic bead tablet 39, which is itself locateddownstream from the branch connection.

[0138] In this position, the volume of air downstream from said testsolution 69 is trapped but remains a significant volume. It is thenpossible, by forcing the air inlet along F4, to compress the trappedair. When several to-and-fro movements are made, the solution 69 willdissolve the magnetic bead tablets 39. These beads will be present in ahomogenous manner in this new solution 70, referred to as the treatedsolution 70, a name which it will be assigned afterwards regardless ofits level or treatment (magnetic beads, structural constituents,functional constituents, amplicons) as soon as the base liquid used isthe test solution 69. In addition, for the wash solution 71 which willbe described below, when this wash solution 71 has fulfilled itsfunction and contains waste that must be discharged, its reference isnot modified. Furthermore, for the elution solution 72 which will alsobe described below, when the elution liquid 72 recovers the structuralconstituents, amplicons or others, this liquid is thus referred to as atreated liquid 70.

[0139] The to-and-fro movement described above, and which will beexplained in association with the wash solution 71 and elution liquid 72below, is possible owing to the configuration of the separationcompartment 38. Thus, this compartment 38 has a greater volume that thevolume of test liquid 69 introduced, such that the liquid 69circumscribes downstream a volume of air which is captured. Owing tothis fact, when said liquid 69 is pushed by an increase in upstreampressure, either by a thrust due to said liquid 69 itself, or by anincrease in the air pressure by the introduction of a surplus gas atinlet 5, and when this thrust is then released, the magnetic bead tablet39 can be driven by a to-and fro movement. This movement thus optimisesthe placement or replacement of said magnetic beads into suspension.

[0140] In this respect, the ratio that exists between the volume of thecompartment 38 upstream of the branch connection 73 and the volume ofsaid compartment 38 downstream from said branch connection 73 is between1:2 and 1:5, and preferably is 1:3. The ratio between the volume of saidsolution 70 and the volume of the compartment 38 downstream of thissolution 70 is thus substantially equal or less. For example, in thecase where this ratio is less, it is between 1:2.5 and 1:6, andpreferably it is 1:3.5. Owing to the ratios mentioned above, in theexample described on the figures, the total volume of the dosingcompartment 36 being 50 μl, the separation compartment 38 is thus 150μl.

[0141] The magnetic beads have the property of binding specificallynucleic acids. To this end, the test solution mixture 69 and themagnetic bead tablets 39 must be subjected to a temperature or atemperature cycle between 25 and 60° C. Then, magnetic separation of thebeads is conducted against at least one of these transparent films 65and/or 66 which partitions the card on its front surface 62 and/or rearsurface 63, at the level of its separation compartment. The magneticseparation of the magnetic beads takes place before the treated solution70 is discharged along F6.

[0142] If required, these treated solution 70 dosing steps and theseparation of nucleic acids can be repeated N times, N being between 1and 10, in order to treat the volumes of treated solution 70 greater tothe volume of the separation compartment 38.

[0143] The magnetic beads are then washed so that only the nucleic acidssought are retained. When the wash solution 71 is introduced along F1,by the inlet 2, the following valves are closed:

[0144] one of the orifices of the first valve 11,

[0145] the inside orifice of the fourth valve 14,

[0146] one of the orifices of the sixth valve 16,

[0147] the inside orifice of the eighth valve 18,

[0148] the inside orifice of the twelfth valve 22,

[0149] one of the orifices of the sixteenth valve 26,

[0150] one of the orifices of the additional valve 61 of the right-handpath,

[0151] the inside orifice of the second valve 12 of the right-hand path,and

[0152] the outside orifice of the fourth valve 14 of the right-handpath.

[0153] The wash solution 71 will thus flow into the separationcompartment 38, where the magnetic beads are still magnetized. Theblocking filter 53 stops this liquid, then the magnetic separation isinterrupted and a to-and-fro movement is performed so that said washsolution 71 can properly wash the nucleic acid-carrying magnetic beads.

[0154] The magnetic separation of magnetic beads is again implementedbefore the wash solution 71 is discharged along F6. The closed valvesare, of course, identical to those used during the discharge of theremaining test solution 70, the nucleic acids of which were captured bythe magnetic beads.

[0155] When the wash solution is reintroduced along F5 to perform asecond washing operation, the same valves as for the first washingoperation are closed, except for the inside orifice of the second valve22 that is open while the inside orifice of the thirteenth valve 23 isclosed. In this configuration, the new wash solution 71 introduced isstopped by the blocking filter 54, connected to the twelfth valve 22.Magnetic separation is interrupted and a to-and-fro movement isconducted so that said liquid 71 can best clean the nucleicacid-carrying magnetic beads.

[0156] Once the washing operation is finished, the wash solution 71present in the channels 64 is replaced by an inert gas, air for example.In this case, air is injected under pressure by the inlet 5 along F4.The discharged wash solution 71 exits the card 1 via the outlet 8 alongF6 through the waste outlet ball valve 10. The closed valves are thusindicated, except for the following characteristics:

[0157] reopening the inside orifice of the first valve 11,

[0158] closure of the inside orifice of the second valve 12,

[0159] closure of the inside orifice of the first valve 11 of theright-hand path, and

[0160] reopening of the inside orifice of the second valve 12 of theright-hand path.

[0161] Of course, this step can be conducted between the two washingsteps.

[0162] This step can be repeated several times, of course, the dirtywash solution 71 always being discharged via the outlet 8, via theoutlet ball valve 10, along F6. In this case, other blocking filtersmust be provided between blocking filters 54 and 55.

[0163] 3^(rd) step-Recovery of magnetic beads by the elution liquid:

[0164] In this third step, the test solution 70 was almost completelydischarged along F6. Only the nucleic acids, which were captured duringthe previous step, remain attached to the magnetic beads. These magneticbeads are still present as magnetic separation is again conducted whilewaiting for the recovery of nucleic acids by an elution liquid 72, whichis the purpose of this step.

[0165] Initially, the elution liquid 72 to be used must be correctlydosed. The volume required in this case is 50 μl. There is thus a ratioof 1:2 between the volume of the treated solution 70 and the volume ofthe elution liquid 72. Nonetheless, a ratio varying between 1:1 and 1:10is possible. Due to the fact that this step can be repeated 1 to 10times, this ratio may thus range from 1 to 100. The volume of elutionliquid 72 is dosed in the dosing compartment 37.

[0166] To do this, the elution liquid 72 is introduced via the inlet 4,along F3. Said liquid 72 then follows the channels 64 to fill the dosingcompartment 37. The liquid 72 is stopped by the blocking filter 50.

[0167] The following valves are closed:

[0168] the inside orifice of the second valve 12,

[0169] the outside orifice of the third valve 13,

[0170] the inside orifice of the sixth valve 16, and

[0171] the outside orifice of the second valve 12 of the right-handpath.

[0172] This configuration is clearly represented in FIG. 5.

[0173] Once the elution liquid 72 dosing operation is finished, it isreplaced in the channels 64 by an inert gaseous fluid, air for example.In this case, air is injected under pressure by the inlet 5 along F4.The discharged elution liquid 72 exits the card 1 via the outlet 8 alongF6 through the waste outlet ball valve 10. A card 1 is thus obtainedwhich has a liquid distribution as represented in FIG. 6. The followingvalves are closed:

[0174] closure of one of the orifices of the first valve 11,

[0175] reopening of the outside orifice of the second valve 12,

[0176] closure of the inside orifice of the eighth valve 18,

[0177] closure of the inside orifice of the fourteenth valve 24,

[0178] closure of the inside orifice of the fifteenth valve 25,

[0179] reopening of the outside orifice but closure of the insideorifice of the third valve 13,

[0180] closure of the inside orifice of the third valve 13 of theright-hand path,

[0181] closure of the inside orifice of the fourth valve 14 of theright-hand path,

[0182] closure of the inside orifice of the eighth valve 18 of theright-hand path,

[0183] closure of the inside orifice of the fourteenth valve 24 of theright-hand path, and

[0184] closure of the inside orifice of the fifteenth valve 25 of theright-hand path.

[0185] The elution liquid 72, which has just been dosed and is presentin the compartment 37, is transferred to the following compartment,referred to as the separation compartment 38. Said liquid 72 located incompartment 36 is thus pushed toward the separation compartment 38, bythe introduction of an inert fluid into the channel network 64 via theinput 5 along F4. The elution liquid 72 is stopped when said liquid 72reaches the blocking filter 55. In this case, the following valves areclosed:

[0186] the inside orifice of the second valve 12,

[0187] the outside orifice of the third valve 13,

[0188] the outside orifice of the ninth valve 19,

[0189] one of the orifices of the fourteenth valve 24,

[0190] the outside orifice of the fifteenth valve 25, and

[0191] the inside orifice of the first valve 11 of the right-hand path.

[0192] The configuration of the card 1 after displacement of the elutionliquid 72 is represented in FIG. 7. In this position, said elutionliquid 72 allows the magnetic beads associated with the nucleic acids tobe recovered. At this stage, it is possible to perform a mixingoperation as indicated above with the test solution 70 and the magneticbeads of the tablet 39.

[0193] The elution liquid 72, which may be a buffer such as Tris at pH7.5 and with weak ionic strength, will enable the nucleic acids of themagnetic beads to be separated, by performing an incubation at atemperature between 25 and 60° C. Magnetic separation of the magneticbeads alone is then conducted, the nucleic acids being in suspension insaid liquid 72.

[0194] 4^(th) step-Transfer if the elution liquid and recovery ofstructural constituents of the amplification:

[0195] This fourth step is represented in FIG. 8. The following valvesare closed:

[0196] the inside orifice of the second valve 12,

[0197] the outside orifice of the third valve 13,

[0198] the outside orifice of the ninth valve 19,

[0199] one of the orifices of the fourteenth valve 24,

[0200] the inside orifice of the fifteenth valve 25,

[0201] the outside orifice of the eighteenth valve 28, and

[0202] the inside orifice of the first valve 11 of the right-hand path.

[0203] In this configuration, the elution liquid 72 containing thenucleic acids extracted from the test solution 70 is transferred fromthe separation compartment 38 to the constituent recovery compartment 40enabling amplification. This concerns structural constituents that allowthis amplification to take place. These constituents are amalgamated andform a tablet 41 located at the entrance of the compartment 40, suchthat the elution liquid will rapidly dissolve the tablet 41 when itenters this compartment 40 and will carry the constituents with it toits position as described in FIG. 8.

[0204] The volume of this compartment 40 is 150 μl, that is with a ratioof 1:3 with the 50 μl of the elution liquid 72. Nonetheless, this ratiocan be 1:2 to 1:5. This ratio allows the displacement of said liquid 72containing the nucleic acids and loaded with structural constituents,which facilitates mixing.

[0205] The transfer is made through the increase in the pressure insidethe card 1 upstream from the elution liquid 72. This pressure increaseis made by injecting air or any other inert gaseous fluid via inlet 5,along F4.

[0206] The liquid 72, containing nucleic acids and containing structuralconstituents of the amplification, will be stopped by the blockingfilter 56 associated with the seventeenth valve 27.

[0207] During this step, the inner orifice of the sixteenth valve 26 andthe inner orifice of the seventeenth valve 27 are closed when saidliquid 72 is in this compartment 40. Incubation in performed for 1 to 15minutes at a temperature between 30 and 65° C., which allows the primersto become attached to the corresponding nucleic acids. Then, thetemperature is adjusted to a value between 37 and 42° C. As describedabove, the liquid derived from this 4^(th) step, which essentiallycontains the elution liquid 72 and the structural constituents likely toallow amplification in certain conditions, is called solution or treatedliquid 70.

[0208] 5^(th) step-Transfer of the treated liquid and recovery of thefunctional constituents of the amplification:

[0209] This fifth step is represented in FIG. 9. The following valvesare closed:

[0210] the inside orifice of the second valve 12,

[0211] the outside orifice of the third valve 13,

[0212] the inside orifice of the ninth valve 19,

[0213] the inside orifice of at least one of the valves 20, 21, 22and/or 23 and/or one of the orifices of the fourteenth valve 24,

[0214] the inside orifice of the fifteen valve 25,

[0215] the inside orifice of the twenty-fourth valve 34, and

[0216] the inside orifice of the first valve 11 of the right-hand path.

[0217] In this configuration, the elution liquid 72, containing thenucleic acids and the structural constituents likely to allowamplification, referred to as the treated solution 70, is transferredfrom the structural constituent recovery compartment 40 allowingamplification to the functional constituent recovery compartment 42enabling this same amplification to take place. The blocking filter 59stops the movement of said treated solution 70.

[0218] These functional constituents essentially contain enzymesallowing the amplification operation to be carried out. The type andnumber of these enzymes and other constituents is a function of theamplification technique chosen. These constituents are amalgamated andform a tablet 43 located at the entrance of the compartment 42, suchthat the solution treated will rapidly dissolve the tablet 43 when itenters this compartment 42 and will carry the constituents with it toits position as described in FIG. 9. This position is only a momentaryintermediate position as the pressure along F4 will allow for thesubsequent transfer of the elution liquid 72 containing the structuraland functional constituents allowing amplification to be performed,according to, of course, another valve configuration.

[0219] The transfer is identical to that of the fourth step. It is thuscarried out by increasing the pressure inside the card 1 upstream fromthe treated liquid 70. Injecting air or any other inert gaseous fluidvia inlet 5, along F4, makes this pressure increase. There is also thesame ration of 1:3 between the liquid volume 70 and the volume of thecompartment 42, with the same to-and-fro mixing effect already describedabove.

[0220] During this step, the inside orifice of the eighteenth valve 28and the inside orifice of the nineteenth valve 29 are closed when saidliquid 70 is in this compartment 42. Heating takes place for 30 to 90minutes at a temperature between 30 and 75° C., preferably between 37and 42° C., if isothermal amplification is desired, using suchtechniques as NASBA, TMA, 3SR, SBA. To perform a PCR, the temperaturesmust be changed several time while remaining within the 30 to 100° C.temperature range, in order to do several successive amplificationcycles.

[0221] It should be noted that the fourth and fifth steps can becombined to form a single step. The only condition is to use stableenzymes at a temperature greater than or equal to 50° C., referred to asthermostable enzymes.

[0222] 6^(th) step-Transfer of the liquid treated into the samplingcompartment, into the detection and reading compartment and into theconvergence compartment:

[0223] During this sixth test, represented in FIGS. 10 to 12, threesuccessive phases occur. These phases are as follows:

[0224] the transfer of an initial part of the treated liquid 70, that issubstantially 20 μl, containing or not containing the amplificationproduct, from the functional constituent recovery compartment 42 to thesampling compartment 44, which corresponds to FIG. 10,

[0225] the transfer of a second part of the treated liquid 70,corresponding to the remainder of said liquid 70, that is substantially30 μl, from the functional constituent recovery compartment 42 to theconvergence compartment 47, which corresponds to FIG. 11, and

[0226] the transfer of this part of said treated liquid 70 from thesampling compartment 44 to the detection and reading compartment 45,which corresponds to FIG. 12.

[0227] According to FIG. 10, the following valves are closed:

[0228] the inside orifice of the second valve 12,

[0229] the outside orifice of the third valve 13,

[0230] the outside orifice of the ninth valve 19,

[0231] the inside orifice of at least one of the valves 20, 21, 22and/or 23 and/or one of the orifices of the fourteenth valve 24,

[0232] the inside orifice of the fifteen valve 25,

[0233] the outside orifice of the twenty-first valve 31,

[0234] the inside orifice of the twenty-fourth valve 34, and

[0235] the inside orifice of the first valve 11 of the right-hand path.

[0236] This transfer allows the precise dosing of an aliquot ofsubstantially 20 μl of treated solution 70, which will then betransferred into the detection and reading compartment 45. During thisphase, the liquid 70 is stopped by a blocking filter 57, at the level ofthe detection and reading compartment 45, and a blocking filter 59,downstream from the convergence compartment 47.

[0237] According to FIG. 11, the closed valves are identical to theprevious phase. Only the following differences are noted:

[0238] opening of the inside orifice of the twenty-fourth valve 34,

[0239] closure of the inside orifice of the twenty-fifth valve 35,

[0240] closure of the outside orifice of the twenty-fourth valve 34 ofthe right-hand path, and

[0241] closure of the inside orifice of the twenty-fifth valve 35 of theright-hand path.

[0242] As such, the transfer to the convergence compartment 47 ispossible for the remainder of the treated liquid 70 which is not takeninto consideration by the compartment 44. The blocking filter 60,associated with the twenty-fifth valve 35, stops this part of saidliquid 70.

[0243] According to FIG. 12, the following valves are closed:

[0244] the inside orifice of the second valve 12,

[0245] the outside orifice of the third valve 13,

[0246] the outside orifice of the ninth valve 19,

[0247] the inside orifice of at least one of the valves 20, 21, 22and/or 23 and/or one of the orifices of the fourteenth valve 24,

[0248] the inside orifice of the fifteen valve 25,

[0249] one of the orifices of the twenty-third valve 33, and

[0250] the inside orifice of the first valve 11 of the right-hand path.

[0251] The nineteenth valve 29 is open, which allows the transfer to thedetection and reading compartment 45 of the liquid 70, previouslycontained in the sampling compartment 44, possibly containing labelledamplicons. It should be noted that during this phase, the aliquot ofsaid liquid 70 will pass through a zone upstream of said compartment 45where a detection tablet 45 a is located. Such a tablet 45 a cancontain, for example, amplicon cutting and labelling products, such asdescribed in patent application PCT/FR99/01469 filed by the applicant onJun. 17, 1999, under priority of Jun. 17, 1998.

[0252] The liquid 70 containing the amplicons possibly labelled andfragmented follows its movement and is stopped in the downstream portionof the compartment 45 by a blocking filter 58, such that said liquid 70is present at the level of a reading cell 46. At this cell 46, it ispossible to check that the amplification has been carried out and thatthe next phase can be performed. This amplification can be checked bymeans of unlabelled oligonucleotides, called capture probes, fixed atthe level of the reading cell 46. These probes are adapted to behybridised with the amplification product which had to be amplified,labelled and fragmented.

[0253] In another embodiment, the liquid 70 containing the unlabelledamplicons is stopped at the level of the cell 46. Amplificationmonitoring implements an unlabelled amplicon detection technique such asthat described in:

[0254] patent application WO-A-95/13399, WO-A-97/39008 or WO-A-99/64432,or patents U.S. Pat. No. 5,283,174, U.S. Pat. No. 5,656,207, U.S. Pat.No. 5,658,737 or U.S. Pat. No. 5,928,862, for similar techniques, and

[0255] in the patent application WO-A-91/19812, or patents U.S. Pat. No.4,889,798 or U.S. Pat. No. 5,998,135, for different techniques.

[0256] 7^(th) step-Transfer of the elution liquid from the convergencecompartment to the outside:

[0257] The aliquot of treated liquid 70 in the convergence compartment47 comes from the left-hand path of the card 1. Nonetheless, anotheraliquot 70, containing or not containing other specific amplicons due toamplification, can also be present coming from the right-hand path ofthe card 1. In the case of FIG. 12, the closed valves are identical buton the opposite paths. It is thus the blocking filter 35 of theright-hand side that enables the arrival of this aliquot to be stoppedin the convergence compartment 47, and thus the mixing of said aliquotsfrom both the right-hand and left-hand paths.

[0258] The amplifications in these two paths can be identical (identicalbuffers used), in order to obtain more amplicons, or different (throughthe use of different buffers), in order to allow several assaysaccording to the number of different amplicons obtained.

[0259] It is also possible to have more than two paths in a reactioncard 1 according to the invention. In this case, the channels 64 must beadapted to the vicinity of the inlet 6 and outlet 9 ball valves. In thisway, it would be necessary to have other blocking filters and othervalves in lower position in order to have all the aliquots converge intothe compartment 47.

[0260] When the reading at the level of the cell 46 proves that theamplification has been carried out correctly, which proves the presenceof a target in the initial biological sample, that is the test solution69, the aliquot(s) present in the convergence compartment 47 are thentransferred to another test sample card or reaction card, not shown inthe figures, which allows the amplicons to be identified and analysed.It can use, for example, DNA chips developed by the Affymetrix Company,as described above. This transfer takes place through outlet 7, alongF5, via the ball valve 9.

[0261] However, it is also possible that a biological chip bestructurally integral with the reaction card 1. It is also possible thatthe sixth stage be limited to the transfer to the convergencecompartment, without there being a control or amplification. Finally,this transfer can be performed directly in the DNA chip without passingthrough the convergence compartment.

REFERENCES

[0262]1. Reaction card

[0263]2. Entrance of the wash solution 71

[0264]3. Entrance of the test solution 69

[0265]4. Entrance of the elution liquid 72

[0266]5. Entrance for pressure variation inside the card 1

[0267]6. Card 1 entrance ball valve

[0268]7. Exit of the treated solution 70 to the outside

[0269]8. Waste outlet

[0270]9. Outside exit ball valve

[0271]10. Waste exit ball valve

[0272]11. First valve

[0273]12. Second valve

[0274]13. Third valve

[0275]14. Fourth valve

[0276]15. Fifth valve

[0277]16. Sixth valve

[0278]17. Seventh valve

[0279]18. Eighth valve

[0280]19. Ninth valve

[0281]20. Tenth valve

[0282]21. Eleventh valve

[0283]22. Twelfth valve

[0284]23. Thirteenth valve

[0285]24. Fourteenth valve

[0286]25. Fifteenth valve

[0287]26. Sixteenth valve

[0288]27. Seventeenth valve

[0289]28. Eighteenth valve

[0290]29. Nineteenth valve

[0291]30. Twentieth valve

[0292]31. Twenty-first valve

[0293]32. Twenty-second valve

[0294]33. Twenty-third valve

[0295]34. Twenty-fourth valve

[0296]35. Twenty-fifth valve

[0297]36. Test solution dosing compartment

[0298]37. Elution liquid dosing compartment

[0299]38. Separation compartment

[0300]39. Magnetic bead tablet

[0301]40. Structural constituent recovery compartment

[0302]41. Tablet of structural constituents enabling amplification totake place

[0303]42. Amplification compartment

[0304]43. Tablet of functional constituents enabling amplification totake place

[0305]44. Sampling compartment

[0306]45. Detection and reading compartment

[0307]45 a. Detection tablet

[0308]46. Reading cell

[0309]47. Convergence compartment

[0310]48. First hydrophobic filter

[0311]49. Second hydrophobic filter

[0312]50. Blocking filter for a determined volume of elution liquid inthe compartment 37

[0313]51. Blocking filter for a determined volume of the solution 70 inthe compartment 36

[0314]52. Blocking filter of the test solution 70 in the compartment 38

[0315]53. Blocking filter of the first wash solution 71 in thecompartment 38

[0316]54. Blocking filter of the second wash solution 71 in thecompartment 38

[0317]55. Blocking filter of the elution liquid in the compartment 38

[0318]56. Blocking filter of the sample with the constituents in thecompartment 40

[0319]57. Blocking filter of the sample in the reading cell 46

[0320]58. Blocking filter of the labelled sample in the compartment 45

[0321]59. Blocking filter of the sample with the enzymes in thecompartment 42

[0322]60. Blocking filter of the sample with the enzymes in thecompartment 47

[0323]61. Additional valve of the right-hand path, referred to as thepurge valve

[0324]62. Front of the card 1

[0325]63. Rear of the card 1

[0326]64. Through channels on the front 62

[0327]65. Transparent film partitioning the channels 64 on the front 62

[0328]66. Transparent film partitioning the channels 64 on the rear 63

[0329]67. Transparent film partitioning the valves 11 to 35 and 61 onthe rear 63

[0330]68. Side or edge of the card 1

[0331]69. Test solution

[0332]70. Treated solution

[0333]71. Wash solution

[0334]72. Elution liquid

[0335]73. Branch connection of the compartment 38 for blocking filters52 to 55

[0336]74. Reinforcement or groove of each valve 11 to 35 and 61 wherethe film 67 is welded

[0337]75. Compression means of the film 67 or flexible tab

[0338]76. Hermetic closure means or elastomer pin

[0339]77. Opening apparatus or wedge

[0340]78. Strip assembly consisting of several tabs 75

[0341]79. Piston type actuator

[0342]80. Compressed air hose

[0343]81. Support

[0344]82. Bevelled surface of the card 1

[0345]83. Blade 78 securing means

[0346]84. Peripheral weld located in the bottom of the groove 74

[0347] F1. Entrance of the wash solution 71

[0348] F2. Entrance of the test solution 70

[0349] F3. Entrance of the elution liquid

[0350] F4. Entrance for pressure variation inside the card 1

[0351] F5. Exit of the test solution to the outside

[0352] F6. Waste outlet

[0353] F7. Inlet of compressed air for actuating means 77

[0354] F8. Outlet of compressed air for actuating means 77

[0355] F9. Movement of actuating means 77

[0356] F10. Tipping of the tab 75

1. A reaction card (1) consisting of a body, having a front surface (62)and a rear surface (63) defined by an edge (68), at least one input (2,3, 4 and/or 5) and at least one output (7 and/or 8), connected one (2,3, 4 and/or 5) to the other (7 and/or 8) through a network of channels(64) constituting at least one reaction path for at least one fluid (70,71 and/or 72), the fluid or fluids being directed inside the card (1)via valves (11 to 35 and/or 61); each valve (11 to 35 or 61) consists ofa flexible film (67), which can be deformed to allow a fluid to passthrough or which cooperates with a compression means (75) to prevent thefluid from passing through, the film (67) being fixed on the rearsurface (63) of said card (1) at the peripheral indentation (74) of theassembly of channels associated with the valve (11 to 35 or 61); thecard (1) features channels (64) flush with at least one of its surfaces(62 and/or 63), the channels (64) being of two different cross-sections,a small cross-section serving as a reaction compartment; each front (62)or rear surface (63) is delimited by at least one film (48, 49, 65, 66and/or 67).
 2. The card, according to claim 1, characterized in that thebody of the card (1) is monobloc, and that the compression means (75) isaffixed and integral with said card (1) or forms a part of an apparatusallowing the card (1) to be implemented.
 3. The card, according toeither of claims 1 or 2, characterized in that the ratio between thesmall cross-section and the large cross-section of the channels (64) isbetween 1:1.01 and 1:10, preferably between 1:1.01 and 1:3.
 4. The card,according to any of claims 1 to 3, characterized in that the channels(64) are flush with all or part of the front surface (62) of the card(1) and the valves are present on the rear surface (63) of said card(1).
 5. The card, according to any of claims 1 to 4, characterized inthat the front surface (62) of the card (1) features a single film (65)at the level of all the channels (64) flush with this surface (62), andthe rear surface (63) of said card (1) features: at least a flexiblefilm (67) on the valves (11 to 35 and/or 61), at least a hydrophobicfilter (48 and/or 49), and possibly at least a film (66) at the level ofthe channels (64) flush with the rear surface (63).
 6. The card,according to claim 5, characterized in that the flexible film (s) (67)and the film(s) (65 and/or 66) make up a single film.
 7. The card,according to any of claims 1 to 6, substantially parallelepiped-shaped,characterized in that the channels (64) are, totally or in part,circumscribed in the middle part of the card (1), that the blockingfilters (50 to 60), associated with the hydrophobic filters (48 and 49),are circumscribed at the level of at least one of the sides of said card(1), and that the valves (11 to 35 and/or 61) are positioned between thechannels (64) and the blocking filters (50 to 60).
 8. The card,according to any of claims 1 to 7, characterized in that the edge (68)includes all of the inlets (2, 3, 4 and 5) and outlets (7 and 8) offluids (70, 71 and 72) of the card (1).
 9. The card, according to claim8, having a substantially parallelepiped shape, characterized in thatthe entry or entries (2, 3, 4 and 5) are presented on one of the sidesforming the edge (68), and that the outlet or outlets (7 and 8) arepresented on another side of this edge (68).
 10. The card, according toclaim 9, characterized in that the inlet or inlets (2, 3, 4 and 5) arepresented on a side opposite the side where the outlet or outlets (7 and8) are presented.
 11. The card, according to claim 10, with asubstantially rectangular parallelepiped shape, characterized in thatthe entry or entries (2, 3, 4 and 5) and the outlet or outlets (7 and 8)are presented on the two sides form the edge (68).
 12. The use of areaction card, according to any of claims 1 to 11, characterized inthat, to test a biological solution (70), possibly pre-treated to freeligands, the various biological steps are conducted in the followingorder: capture of ligands, recovery of captured ligands in an elutionsolution (72), mixture of recovered ligands with structural andfunctional constituents allowing the ligands to be treated, andqualitative and/or quantitative detection of treated ligands.
 13. Theuse of a reaction card, according to any of claims 1 to 11,characterized in that, to test a biological solution (70), possiblypre-treated to free nucleic acids, the various biological steps areconducted in the following order: capture of nucleic acids, recovery ofcaptured nucleic acids in a elution solution (72), mixture of nucleicacids recovered with the structural and functional constituents allowingamplification of these nucleic acids, and qualitative and/orquantitative detection of amplification products.
 14. The use, accordingto either claims 12 or 13, characterized in that a last step isperformed consisting in analysing these ligands or these amplificationproducts, either in a new location within the card (1), or aftertransmission to another apparatus.
 15. The use, according to any ofclaims 12 to 14, characterized in that the ligands or the capturednucleic acids are subjected to at least a wash solution (71) prior totheir recovery.
 16. The use, according to claim 15, characterized inthat the capture and possible washing operations are conducted N timesin succession, N being between 1 and
 10. 17. The use, according toeither claim 12 or 13, characterized in that a treatment of ligands oran amplification of nucleic acids is conducted prior to the transmissionto a compartment of the card (1) or to another apparatus allowing theligands or the amplicons to be analyzed.
 18. The use, according to anyof claims 12 to 16, characterized in that the card (1) is used after anapparatus enabling a test solution (69) to be treated, such as anapparatus designed to lyse biological cells and to free the ligands,such as nucleic acids, and before an apparatus designed to analyse thepresence of these ligands, such as a DNA chip.